Silver nanoparticle-loaded chitosan–starch based films: Fabrication and evaluation of tensile, barrier and antimicrobial properties

Materials Science and Engineering C 30 (2010) 891–897

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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c

Silver nanoparticle-loaded chitosan–starch based films: Fabrication and evaluation of tensile, barrier and antimicrobial properties Rangrong Yoksan a,⁎, Suwabun Chirachanchai b a b

Department of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart University, 50 Paholyothin Rd., Ladyao, Jatujak, Bangkok 10900, Thailand The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand

a r t i c l e

i n f o

Article history: Received 18 December 2009 Received in revised form 6 March 2010 Accepted 7 April 2010 Available online 21 April 2010 Keywords: Silver nanoparticles γ-Ray irradiation Chitosan Starch Antimicrobial film

a b s t r a c t The fabrication of silver nanoparticles was accomplished by γ-ray irradiation reduction of silver nitrate in a chitosan solution. The obtained nanoparticles were stable in the solution for more than six months, and showed the characteristic surface plasmon band at 411 nm as well as a positively charged surface with 40.4 ± 2.0 mV. The silver nanoparticles presented a spherical shape with an average size of 20–25 nm, as observed by TEM. Minimum inhibitory concentration (MIC) against E. coli, S. aureus and B. cereus of the silver nanoparticles dispersed in the γ-ray irradiated chitosan solution was 5.64 µg/mL. The silver nanoparticle-loaded chitosan– starch based films were prepared by a solution casting method. The incorporation of silver nanoparticles led to a slight improvement of the tensile and oxygen gas barrier properties of the polysaccharide-based films, with diminished water vapor/moisture barrier properties. In addition, silver nanoparticle-loaded films exhibited enhanced antimicrobial activity against E. coli, S. aureus and B. cereus. The results suggest that silver nanoparticle-loaded chitosan–starch based films can be feasibly used as antimicrobial materials for food packaging and/or biomedical applications. © 2010 Elsevier B.V. All rights reserved.

1. Introduction For decades, silver nanoparticles have been widely used as antimicrobial agents in a number of areas, including dental [1,2], medical and pharmaceutical [3–9], textile and fiber [10–16], coating and paint [17,18], film [19], membrane [20] and food packaging purposes [21]. Conventionally, silver nanoparticles are produced by the reduction of silver salt precursors using chemical reducing agents in the presence of stabilizers. Although chemical reduction is an easy and convenient method, chemical reducing agents such as NaBH4 [22], formamide [23], dimethylformamide [23,24], triethanolamine [23], hydrazine [25], etc., are involved in the production. The removal of these reducing agents is cost- and time-intensive and their residues are toxic. The reduction of silver salts by γ-ray irradiation [26–29], microwave irradiation [30–32], photochemical process [33,34] and sonochemical process [35] has been reported to produce metal nanoparticles without, or with fewer, chemical concerns. Previously, we studied the effects of γ-ray doses (2.5–25.0 kGy) as well as concentrations of silver nitrate salt precursor (0.02–0.10 mmol) and chitosan solution (0.1 and 0.5% w/v) on the size and number of the formed silver nanoparticles (Ag0) [36]. However, the antimicrobial activities of the obtained nanoparticles dispersed in γ-ray irra-

⁎ Corresponding author. Tel.: + 66 2 562 5097; fax: + 66 2 562 5092. E-mail address: [email protected] (R. Yoksan). 0928-4931/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.04.004

diated chitosan and their feasible application as antimicrobial agents for food packaging and biomedical devices have not been elaborated. The application of silver nanoparticles in antimicrobial food packaging is possible, as revealed in previous reports [21,37,38]. Many efforts have been made to load and/or to incorporate silver nanoparticles into acceptable packaging materials such as filter paper [21], low density polyethylene (LDPE) [37], and poly(methyl methacrylate) (PMMA) [38]. Also, numerous biodegradable materials, e.g. poly(vinyl alcohol) [39] and polysaccharides such as starch [40], chitosan [19,41– 43], alginate [43] and konjak glucomannan [33] have been used to fabricate silver nanoparticle-based composite films. Starch and chitosan are naturally abundant polysaccharides which are generally non-toxic, available from renewable agricultural sources, and suitable for film formation. Although starch film is cheap and easily biodegradable, it is very sensitive to moisture and exhibits poor mechanical properties. Chitosan possesses many unique properties, including antimicrobial characteristics; hence it has been used in various applications, such as medical, pharmaceutical, textile, water treatment, food, cosmetics, packaging, etc. [44]. The blending of starch and chitosan is an alternative way to not only improve the mechanical and water vapor barrier properties, as well as the antimicrobial attributes, of starch film [45–48], but also to reduce the cost and enhance the biodegradability of chitosan film [48]. The aims of the present research are thus to determine the minimum inhibitory concentration (MIC) of silver nanoparticles dispersed in chitosan solution, and to investigate the effects of silver nanoparticle content on the tensile properties, the water vapor and oxygen

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gas barrier properties, and the antimicrobial qualities of silver nanoparticle-loaded chitosan–starch based films. 2. Experimental 2.1. Materials Chitosan (deacetylation degree of 0.95 and molecular weight of ∼ 700,000 Da) was purchased from Seafresh Chitosan (Lab) Co. Ltd., Thailand. Rice flour and waxy rice flour were acquired from Cho Heng Rice Vermicelli Factory Co. Ltd., Thailand. Silver nitrate and acetic acid were supplied by Merck, Germany. Magnesium nitrate was obtained from Ajax Finechem Pty. Ltd., Australia. Mueller-Hinton broth was purchased from Becton Dickinson, USA. Nutrient broth was supplied by HiMedia Laboratories Pvt. Ltd., India. The agar powder was a product of Purified Agar Co. Ltd., Thailand. The glycerol applied was of commercial grade. All chemicals were used as delivered and without any further purification.

i.e. rice starch (RS) and waxy rice starch (WRS) (2.0% w/v), were prepared by agitation of starches in water: at 100 °C for 60 min and 85 °C for 40 min for RS and WRS, respectively. Chitosan, RS and WRS solutions were mixed together at 50 °C to obtain a mixture with a weight ratio of chitosan to RS to WRS of 2:1:1. Glycerol (20% w/w), γ-ray irradiated chitosan solution, and γ-ray irradiated chitosan solution containing silver nanoparticles were then individually added to samples of the mixture. Different amounts of those components were used to prepare four types of film, as tabulated in Table 1. The homogeneous mixtures were poured onto acrylic plates (31 × 31 cm2) and dried in a hot air oven at 50 °C overnight. The obtained films were peeled from the plates, neutralized in a closed chamber containing ammonia solution (25% v/v), thoroughly washed with water and dried at an ambient temperature (25 °C). Neat chitosan and starch films were also prepared by a solution casting method as described above and used as controls for the antimicrobial activity test. Chitosan film was fabricated from 544 mL of chitosan solution (1.2% w/v), while starch film was produced from 163 mL of each of RS and WRS solutions (2.0% w/v).

2.2. Preparation of silver nanoparticles Silver nanoparticles dispersed in chitosan solution were prepared according to a γ-ray irradiation reduction method described previously [36]. Briefly, chitosan solution (0.5% w/v) was prepared by stirring chitosan flakes in an aqueous acetic acid solution (1% v/v) at an ambient temperature overnight. Freshly prepared silver nitrate (AgNO3) solution (50 mM, 0.04 mmol, 0.8 mL) and aqueous acetic acid solution (1% v/v, 1.2 mL) were then added to the chitosan solution (20 mL). The homogeneous mixture was irradiated by γ-rays with a dose of 25 kGy in a 60Co Gammacell irradiator (Best Theratronics Ltd., Canada) at a dose rate of 12 kGy/h. 2.3. Characterization of silver nanoparticles The UV–Vis absorption spectrum of the obtained particles dispersed in γ-ray irradiated chitosan solution was recorded over a wavelength range from 300 to 500 nm using a Helios Gamma spectrometer (Thermo Scientific, UK). Zeta potential and particle size were determined at 20 °C using a Zetasizer 3600 (Malvern Instruments Ltd., UK) equipped with a He–Ne laser operating at 4.0 mW and 633 nm with a fixed scattering angle of 90°. Transmission electron microscopy (TEM) analysis was performed using a Hitachi H-7650 (Hitachi HighTechnologies Corp., Japan) at an accelerating voltage of 100 kV.

2.6. Study of tensile properties of silver nanoparticle-loaded chitosan– starch based films A film sample (2.5 ×15 cm2) was preconditioned in a closed chamber containing saturated magnesium nitrate solution (Mg(NO3)2) at an ambient temperature (∼50 ± 2% RH) for 2 days. Tensile testing was performed with a universal testing machine (model 1000, Instron, USA) according to the ASTM D882-91, with a crosshead speed of 50 mm/min and a grip separation of 100 mm. For each sample, 8–10 specimens were tested and the tensile properties (tensile strength, modulus of elasticity and elongation at break) were reported as mean ± SD. Tensile strength was calculated by dividing the maximum load by the initial cross-sectional area of the specimen. Modulus of elasticity was determined by extending the linear portion of the load–extension curve and dividing the difference in stress corresponding to any segment of section on this straight line by the corresponding difference in strain. The tensile strength and elastic modulus were expressed in MPa. Elongation at break was reported as a percentage and calculated by dividing the extension length (change in length) at the point of specimen rupture by the initial length of the specimen and multiplying by 100. 2.7. Study of barrier properties of silver nanoparticle-loaded chitosan– starch based films

2.4. Study of antimicrobial activity of silver nanoparticles Minimum inhibitory concentration (MIC) was determined using a tube dilution method. Three strains of bacteria — a Gram-negative bacterium, Escherichia coli (E. coli, ATCC35218), and two Gram-positive bacteria, Staphylococcus aureus (S. aureus, ATCC6538) and Bacillus cereus (B. cereus, ATCC11778) — were applied as test organisms. Serial dilutions were made of the samples in Mueller-Hinton broth (MHB) which was used as a bacterial growth medium. The test organisms in MHB (106 CFU/mL, 1 mL) were then added to those sample dilutions (1 mL). The mixtures (2 mL) were homogeneously mixed using a vortex, subsequently incubated at an ambient temperature for 24 h, and then scored for growth. The MIC was defined as the lowest concentration resulting in the lack of visible growth of microorganisms. 2.5. Preparation of silver nanoparticle-loaded chitosan–starch based films Silver nanoparticle-loaded chitosan–starch based films were prepared by a solution casting method. Chitosan flakes were dissolved in aqueous acetic acid solution (1% v/v) by stirring overnight to obtain a homogeneous chitosan solution (1.2% w/v). Two solutions of starches,

Water vapor transmission rate (WVTR) was evaluated by a Gravimetric Modified Cup Method based on ASTM E96 [48,49]. A film sample with a diameter of 7.5 cm was covered and sealed to the open mouth of a test cup, with an inner diameter of 6.3 cm, which contained dried desiccants (25 mL). The assembly was weighed and

Table 1 Components and their contents used for preparation of chitosan–starch based film (A) and silver nanoparticle-loaded chitosan–starch based films (B–D). Component

Film type A

B

C

D

γ-Ray irradiated chitosan solution (0.5% w/v) 0.0 26.6 53.2 106.4 containing silver nanoparticles (mL)a,b 79.8 53.2 0.0 γ-Ray irradiated chitosan solution (0.5% w/v) (mL)a 106.4 Chitosan solution (1.2% w/v) (mL)a 250 250 250 250 Rice starch solution (2.0% w/v) (mL)c 75 75 75 75 Waxy rice starch solution (2.0% w/v) (mL)c 75 75 75 75 Glycerol (g) 1.2 1.2 1.2 1.2 a b c

1% (v/v) aqueous acetic acid solution was used as a solvent. Concentration of silver nanoparticles was 0.1806 mg/mL. Water was used as a solvent.

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placed in a controlled atmosphere of 25 °C and 50% RH. The test unit was periodically removed and weighed once an hour for the first day, and after that once a day for the duration of 7 days. The weight increment as a function of time was recorded. WVTR was taken as the slope of the curve (in the linear region) divided by the area of the cup opening. For each sample, 6 specimens were tested and the results were reported as mean ± SD. Oxygen transmission rate (OTR) was evaluated according to the ASTM D3985-02 at 23 °C and 0% RH using an Illinois 8000 oxygen permeation tester (Illinois Instruments Inc., USA). For each sample, 3 specimens were tested and the results were reported as mean ± SD. 2.8. Contact angle measurement of silver nanoparticle-loaded chitosan– starch based films Contact angle measurement was performed according to a method modified from the one described by Suyatma et al. [50]. A test film was mounted on a sample stage and leveled horizontally. A drop of distilled water (5 µL) was placed on the surface of the film using a Gastight 500 µL precision syringe (Hamilton Co., USA). Images of the water droplet were recorded within 5 s. The contact angle of the water droplet in air on the surface of the film was then measured by a Dataphysics OCA 15EC (Dataphysics Instruments GmbH, Germany). The contact angles were measured on both sides of the film and averaged. Five measurements were carried out on each side. 2.9. Study of antimicrobial activity of silver nanoparticle-loaded chitosan–starch based films Film samples, each with a diameter of 28 mm, were pasteurized in an autoclave at 121 °C for 15 min. Antimicrobial activity of the film was investigated by an agar disc diffusion method. Three bacterial strains, E. coli, S. aureus and B. cereus, were used as test organisms. Bacterial suspensions with a turbidity equivalent to a McFarland 0.5 standard were prepared (108 CFU/mL) and then diluted to 105 CFU/ mL with nutrient broth (NB). The adjusted bacterial suspensions (0.1 mL) were spread onto nutrient agar plates, which contained a mixture of NB (0.8% w/v), agar powder (1.5% w/v, a solidifying agent) and distilled water. Subsequently, the film discs were placed in direct contact with the agar medium. Plates were inverted and incubated at 37 °C for 24 h. The diameters of clear inhibition zones, including the diameter of the disc (mm), were measured using a Vernier caliper. The measurement was done in triplicate for each sample.

Fig. 1. Physical appearances of (a) γ-ray irradiated chitosan solution and (b) γ-ray irradiated chitosan solution containing AgNO3.

of the solution was more intense in the presence of silver nitrate, thus implying the formation of silver particles (Ag0). The successful fabrication of silver particles was confirmed by UV–Vis spectroscopy from the appearance of the maximum absorption band at 411 nm (Fig. 2) which corresponds to the characteristic surface plasmon band of silver nanoparticles, as reported elsewhere [51,52]. The concentration of the metal particles in γ-ray irradiated chitosan solution was 0.1806 mg/mL, as determined by UV–Vis spectroscopy (see Supplementary data). The individual silver particles were spherical with a mean diameter of ∼20–25 nm, as observed by TEM (Fig. 3). However, dynamic light scattering (DLS) technique showed relatively larger particle size, i.e. 46.7 ± 3.5 nm (Fig. 4a), as compared to the diameters determined by TEM. The larger particle size might be a result of particle aggregation and/or a thin chitosan layer coating the individual particles. Fig. 4a reveals the unimodal particle size distribution of those nanoparticles and/or nanoaggregates. The nanoparticles exhibited a positively charged surface with a zeta potential of 40.4 ± 2.0 mV (Fig. 4b). The positive surface charge might be imparted by protonated amino groups (–NH+ 3 ) of chitosan molecules surrounding the particles. This would support the above assumption, i.e. the surfaces of the silver nanoparticles were coated with a chitosan layer. The high value of zeta potential suggests the stability of the silver nanoparticles in γ-ray irradiated chitosan solution. The nanoparticles were stable in the solution for more than 6 months without tendency to precipitate. The stability of the particles might be a result of electrostatic repulsion.

3. Results and discussion 3.1. Characteristics of silver nanoparticles According to our previous report [36], silver nanoparticles were prepared by the γ-ray irradiation reduction method using silver nitrate as a salt precursor and chitosan as a stabilizer. The concentration of chitosan solution (0.1 and 0.5% w/v), silver nitrate content (0.02, 0.04, 0.06, 0.08 and 0.10 mmol) and γ-ray dose (2.5, 5.0, 10.0, 15.0, 20.0 and 25.0 kGy) were varied in order to determine the optimal reduction condition. The criteria to select the metal nanoparticle fabrication condition are high particle production yield and small sized particles with narrow size distribution and good dispersion (low aggregation and/or agglomeration of the obtained nanoparticles). It was found that the preparation condition using chitosan solution concentration of 0.5% w/v, silver nitrate content of 0.04 mmol and γ-ray dose of 25 kGy was one of the optimal conditions that met those criteria and thus used to fabricate silver nanoparticles for the present study. After γ-ray irradiation (25 kGy), the chitosan solution (0.5% w/v) turned yellow (Fig. 1a), while the chitosan solution containing AgNO3 (0.04 mmol) became red-brown (Fig. 1b). In other words, the color

Fig. 2. UV–Vis spectrum of γ-ray irradiated chitosan solution containing AgNO3.

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Fig. 3. TEM micrographs at 100 kV of silver nanoparticles.

3.2. Antimicrobial activity of silver nanoparticles Minimum inhibitory concentration (MIC) was determined in order to assess the antimicrobial activity of the γ-ray irradiated chitosan solution containing silver nanoparticles. The MICs of acetic acid solution and γ-ray irradiated chitosan solution were also comparatively investigated. In general, MHB culture medium is transparent and colorless; however, with the addition of bacterial dispersions and subsequent incubation at an ambient temperature for 24 h, the medium became turbid (data not shown), indicating bacterial growth. The turbidity of the mixtures decreased with increasing sample concentration (Fig. 5). In other words, the mixtures were more transparent when higher sample concentrations were used, reflecting the suppression of bacterial growth (Fig. 5, left tubes). Similarly, a decrease in transparency or an increase in turbidity of the mixtures was

Fig. 4. (a) Size and (b) zeta potential and their distributions of silver nanoparticles dispersed in chitosan solution (n = 10).

Fig. 5. Appearances of culture mixtures containing different bacterial strains and samples with various concentrations after incubation at ambient temperature for 24 h. Bacterial strains: (1) E. coli, (2) S. aureus and (3) B. cereus. Samples: (A) acetic acid solution, (B) γ-ray irradiated chitosan solution and (C) γ-ray irradiated chitosan solution containing silver nanoparticles. Sample concentrations (μg/mL) — for (A): (a) 2625.00, (b) 1312.50, (c) 656.25, (d) 328.13, (e) 164.06, (f) 82.03, (g) 41.02 and (h) 20.51; for (B): (a) 1250.00, (b) 625.00, (c) 312.50, (d) 156.25, (e) 78.13, (f) 39.06, (g) 19.53 and (h) 9.77; for (C): (a) 45.15, (b) 22.57, (c) 11.29, (d) 5.64, (e) 2.82, (f) 1.41, (g) 0.71 and (h) 0.35.

observed in the tubes with reduced sample concentrations, implying bacterial growth (Fig. 5, right tubes). As Fig. 5A clearly shows, the MICs of acetic acid solution against E. coli, S. aureus and B. cereus are 626.25, 626.25 and 1312.50 μg/mL, respectively. This result suggested that acetic acid solution could inhibit bacterial growth; however, the growth inhibition strength against E. coli and S. aureus was greater than that against B. cereus. γ-Ray irradiated chitosan solution possessed MICs of 312.50, 312.50 and 625.00 µg/mL against E. coli, S. aureus and B. cereus, respectively (Fig. 5B). The antimicrobial activity of the γ-ray irradiated chitosan solution was about two times superior to that of acetic acid solution. Furthermore, the MIC of γ-ray irradiated chitosan solution containing silver nanoparticles decreased to 5.64 µg/mL for all test bacteria (Fig. 5C), reflecting the strongest antimicrobial activity among the three tested samples. This would seem to indicate a direct effect of the silver nanoparticles. The growth inhibition efficiency of silver nanoparticles against all test bacteria was not significantly different. In light of previous

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reports, it should be noted that MIC of the silver nanoparticles depended on the preparation conditions and methods applied. For example, silver nanoparticles synthesized by an electrochemical method exhibited MICs against E. coli and S. aureus of 5 and 2 µg/mL, respectively [53]; while nanoparticles devised by the application of Phytophthora infestans showed very low MICs of 0.313 and 0.625 µg/ mL against E. coli and S. aureus, respectively [54]. In contrast, nanoparticles prepared by the process involving photoassisted reduction gave relatively higher MIC values: 6.25 and 12.5 µg/mL against E. coli and S. aureus, respectively [55]. The difference in MIC values might be a result of the variation of nanoparticle size and shape. 3.3. Characteristics and tensile properties of silver nanoparticle-loaded chitosan–starch based films Four chitosan–starch based films were prepared by varying the volume of γ-ray irradiated chitosan solution containing silver nanoparticles to obtain films with silver nanoparticle contents of 0 (film A), 0.07 (film B), 0.15 (film C) and 0.29% (w/w) (film D) (Table 1). Without silver nanoparticle incorporation, film A (control) was transparent and slightly yellowish, while silver nanoparticle-loaded chitosan– starch based films (films B–D) were pale brown. Color intensity of the film increased with the content of silver nanoparticles. Tensile strength (TS), modulus of elasticity, and elongation at break (E) of the films are tabulated in Table 2. Film A showed TS and modulus of 67 and 5719 MPa, respectively, while the silver nanoparticle-loaded films (films B–D) gave TS in a range from 70 to 75 MPa, and a modulus ranging from 6398 to 6511 MPa. Table 2 shows that TS of the films containing silver nanoparticles of 0.07–0.15% (w/w) (films B–C) is not markedly different from the control film (film A); while the film with silver nanoparticle content of 0.29% (w/w) (film D) exhibited superior TS. The slight increase in TS of chitosan film by incorporation of silver nanoparticles is in agreement with the report of Rhim et al. [19]. The modulus of the films containing silver nanoparticles differed significantly from that of the control film. Although the value tended to increase with an increased content of silver nanoparticles, statistical analysis shows an insignificant difference among the silver nanoparticle-loaded films. In addition, the incorporation of silver nanoparticles resulted in a slight increase in E from 4.60 (film A) to a value ranging from 6.46 to 7.66% (films B–D) (Table 2). The E of the films containing silver nanoparticles of 0.15–0.29% (w/w) was significantly different from that of the control. The tensile results suggested that the incorporation of silver nanoparticles could somewhat improve the tensile properties of chitosan–starch based film. 3.4. Barrier properties of silver nanoparticle-loaded chitosan–starch based films The linear increment of the weight change of desiccants during storage at 25 °C and 50% RH reflected the same water vapor and/or moisture transmission rate of the films over a test period (data not shown). The water vapor transmission rate (WVTR) of film A was 47.6 g m− 2 day− 1; the addition of silver nanoparticles resulted in a

Table 2 Tensile strength, modulus and elongation at break of chitosan–starch based film (A) and silver nanoparticle-loaded chitosan–starch based films (B–D). Film Thickness type (mm)

Tensile strength (MPa)

Modulus (MPa)

Elongation at break (%)

A B C D

66.814 ± 3.269b 69.601 ± 4.518ab 68.948 ± 11.408ab 74.554 ± 5.565a

5719.25 ± 418.87b 6398.46 ± 248.72a 6432.72 ± 982.98a 6511.43 ± 374.45a

4.598 ± 1.048b 6.463 ± 2.413ab 7.230 ± 2.394a 7.655 ± 2.394a

0.040 ± 0.004 0.032 ± 0.004 0.032 ± 0.004 0.034 ± 0.005

Mean values in the same column with different superscripts are significantly different (p b 0.05 using Duncan's multiple range test).

895

Table 3 Water vapor transmission rate (WVTR) and oxygen transmission rate (OTR) of chitosan– starch based film (A) and silver nanoparticle-loaded chitosan–starch based films (B–D). Film type

Water vapor transmission rate

Oxygen transmission rate

Thickness (mm)

WVTR (g m− 2 day− 1)

Thickness (mm)

OTR (cm3 m− 2 day− 1)

A B C D

0.035 ± 0.000 0.039 ± 0.002 0.042 ± 0.003 0.031 ± 0.003

47.60 ± 0.36c 47.75 ± 1.18c 55.76 ± 2.75b 59.21 ± 2.10a

0.039 ± 0.002 0.041 ± 0.003 0.044 ± 0.002 0.038 ± 0.002

2.39 ± 0.02a 1.97 ± 0.01b 1.60 ± 0.01c 1.48 ± 0.03d

Mean values in the same column with different superscripts are significantly different (p b 0.05 using Duncan's multiple range test).

slight increase, to 47.8–59.2 g m− 2 day− 1 (films B–D) (Table 3). In addition, WVTR tended to increase with the augmentation of silver nanoparticle content. The rise in WVTR might be a result of the obstruction of intermolecular hydrogen bond formation between chitosan–chitosan, chitosan–starch and starch–starch molecules by silver nanoparticles, causing the incompatibility of the film matrix, adsorption of water vapor at the hydrophilic sites of polysaccharide molecules, and the eventual penetration of moisture. The result implied that the incorporation of silver nanoparticles suppressed barrier properties against water vapor and/or moisture of the chitosan–starch based film. This result was in agreement with that reported by Shelma et al. [56]. Their result showed that WVTR of chitosan-based film rose slightly due to increased amount of reinforcement, i.e. chitin whiskers. In contrast, films B–D, which contained silver nanoparticles, showed a lower oxygen transmission rate (OTR) (1.48–1.97 cm3 m− 2 day− 1) than film A (2.39 cm3 m− 2 day− 1) (Table 3). The OTR tended to fall with an increasing content of silver nanoparticles. The reduction of OTR might be due to the increment of diffusion path length; as a result the traveling of gas molecules through the matrix was retarded. The result suggested that silver nanoparticles enhanced oxygen barrier properties of the chitosan–starch based film. 3.5. Contact angle of silver nanoparticle-loaded chitosan–starch based films Wettability and hydrophobic characteristics of the materials can be determined from the contact angle of water. A lower contact angle is generally observed for less hydrophobic materials or the materials with good water wettability. Film A showed a contact angle of 95.6°, while films B–D gave lower contact angles, i.e. in the range of 81.5– 88.1° (Fig. 6). This implied that the incorporation of silver nanoparticles increases wettability of the chitosan–starch based film. The decrease in water contact angle of the nanoparticle-loaded films might be a result of the reduced interaction of the film matrix caused by the silver nanoparticles, as mentioned above, resulting in a lower surface tension of the matrix. This information supported the WVTR results. Water was thus more accessible to the surfaces of the silver nanoparticle-loaded films (films B–D) as opposed to that of the control film (film A), leading to a higher level of WVTR for the nanoparticle-loaded films. 3.6. Antimicrobial activity of silver nanoparticle-loaded chitosan–starch based films Antimicrobial activity of the films was investigated by an agar disc diffusion method using three strains of bacteria: E. coli, S. aureus and B. cereus. The inhibitory effect of the films without (film A) and with silver nanoparticles (films B–D) against those bacteria is shown in Fig. 7. After incubation at 37 °C for 24 h, the growth of bacteria underneath film A was observed and bacteria approached the rim of the film (Fig. 7a). In the case of films incorporating silver nanoparticles from 0.07 to 0.29% (w/w), the agar surface in contact with the

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R. Yoksan, S. Chirachanchai / Materials Science and Engineering C 30 (2010) 891–897 Table 4 Antimicrobial activity against E. coli, S. aureus and B. cereus of chitosan–starch based film (A) and silver nanoparticle-loaded chitosan–starch based films (B–D). Sample

Starch film Chitosan film Film A (control) Film B Film C Film D a b

Fig. 6. Appearances of water drops on different film surfaces: (a) film A (θ = 95.58 ± 1.45°), (b) film B (θ = 88.09 ± 3.35°), (c) film C (θ = 84.91 ± 2.63°) and (d) film D (θ = 81.52 ± 3.83°) (n = 6).

films was transparent, indicating no growth of bacteria (Fig. 7b–d). In addition, clear inhibitory zones were observed for the films containing silver nanoparticles. These results suggested that silver nanoparticleloaded chitosan–starch based films (films B–D) exhibited antimicrobial activity against all test bacteria. Table 4 shows that a clear inhib-

E. coli

S. aureus

B. cereus

Inhibitorya

Contactb

Inhibitory

Contact

Inhibitory

Contact

0 28 28

+ − ±

0 28 28

+ − ±

28 28

+ − ±

32.0 ± 0.5 32.6 ± 0.5 33.3 ± 0.5

− − −

30.0 ± 0.6 32.0 ± 0.0 32.3 ± 0.5

− − −

30.3 ± 0.5 31.5 ± 0.6 31.8 ± 0.4

− − −

Inhibitory zone, measured diameter in mm. Contact area under film discs on agar surface; + : growth in the area, − : no growth.

itory zone was not observed for starch film, and that the agar surface in contact with the film was totally turbid, implying the growth of bacteria on the starch film. For chitosan film, although a clear zone surrounding the film disc was not visible, the contact area was transparent, reflecting the antimicrobial activity of chitosan which has been previously reported [57]. The antimicrobial activity of starch film was improved by blending starch with chitosan. Film A (control film) showed an inhibitory area of 28 mm, which corresponded to the area of the film. It should be noted that an inhibitory area larger than 28 mm reflected antimicrobial efficiency. The inhibitory areas of silver nanoparticle-loaded chitosan–starch based films (B–D) were in the range of 30.0–32.3, 30.3–31.8 and 32.0–33.3 mm for S. aureus, B. cereus and E. coli, respectively, implying antimicrobial activities of these films. The growth inhibition of the films against the Gram-negative bacterium E. coli was greater than that against the Gram-positive bacteria S. aureus and B. cereus. This might be a result of the difference in cell wall structure of those microorganisms, especially a possession of the outer membrane (a negatively charged lipopolysaccharide layer) and a thickness of the peptidoglycan layer [58]. The Gram-negative bacteria possess a negatively charged outer membrane and a thin peptidoglycan layer (∼ 7–8 nm), which facilitated the anchoring and penetrating of the silver nanoparticles [59]. In contrast, the Grampositive bacteria lack the outer membrane and have only a thick three dimensional rigid structured peptidoglycan layer (∼ 20–80 nm), which limited the anchoring and penetrating of the positively charged silver nanoparticles [59]. The clear inhibitory zone was larger when silver nanoparticle content increased, implying that the nanoparticles affected the antimicrobial activities of the films. Several antimicrobial mechanisms of the silver nanoparticles have been proposed. Sui et al. revealed that the positive charges available on the silver nanoparticle surface participated in the antimicrobial activity [59]. Nevertheless, Kim et al. reported that the free radicals formed at the surface of silver nanoparticles induced membrane damage [58]. The nanoparticles may attach to the surface of the cell membrane and penetrate inside the bacterial cell, thus disturbing permeability and respiration functions of the cell [60–62] and modulating tyrosine phosphorylation of putative peptide substrates critical for cell viability and division [63]. In addition, Sondi and Salopek-Sondi disclosed that the formation of “pits” in the cell wall of bacteria and accumulation of the silver nanoparticles in the bacterial membrane caused the permeability, resulting in cell death [64]. 4. Conclusion

Fig. 7. Inhibitory zones of different sample films against different bacterial strains after incubation at 37 °C for 24 h. Sample films: (a) film A and (b) film D. Bacterial strains: (A) E. coli, (B) S. aureus and (C) B. cereus.

Silver nanoparticles were successfully synthesized by 25 kGy γ-ray irradiation of 0.04 mmol silver nitrate in 0.5% (w/v) chitosan solution, as confirmed by the appearance of the characteristic surface plasmon band at 411 nm. The obtained silver nanoparticles were highly stable in γ-ray irradiated chitosan solution. They possessed a spherical shape with an average size of 20–25 nm, and exhibited a positively charged surface (40.4 ± 2.0 mV). γ-Ray irradiated chitosan solution containing

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silver nanoparticles showed MIC against E. coli, S. aureus and B. cereus of 5.64 µg/mL. The incorporation of silver nanoparticles into chitosan– starch based films led to a slight improvement of tensile and oxygen gas barrier properties of the films. However, water vapor/moisture barrier properties of the polysaccharide blend films were inferior when loaded with silver nanoparticles. In addition, the silver nanoparticle-loaded chitosan–starch based films exhibited bactericidal performance against E. coli, S. aureus and B. cereus, as demonstrated by the clear inhibitory zone surrounding the films. Acknowledgements This work was financial supported by the Thailand Research Fund (Grant No. MRG5080398) and KU-Institute for Advanced Studies, Kasetsart University. The authors thank the Office of Atoms for Peace, Ministry of Science and Technology, Thailand, for γ-ray irradiation using a 60Co Gammacell irradiator. Appreciation is also expressed to Hitachi High-Technologies Corporation, Japan, for TEM observation. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.msec.2010.04.004. References [1] M.Z. Kassaee, A. Akhavan, N. Sheikh, A. Sodagar, J. Appl. Polym. Sci. 110 (2008) 1699. [2] S.-J. Ahn, S.-J. Lee, J.-K. Kook, B.-S. Lim, Dent. Mater. 25 (2009) 206. [3] F. Furno, K.S. Morley, B. Wong, B.L. Sharp, P.L. Arnold, S.M. Howdle, R. Bayston, P.D. Brown, P.D. Winship, H.J. Reid, J. Antimicrob. Chemother. 54 (2004) 1019. [4] U. Samuel, J.P. Guggenbichler, Int. J. Antimicrob. Agents 23 (SUPPL. 1) (2004) S75. [5] Y. Li, P. Leung, L. Yao, Q.W. Song, E. Newton, J. Hosp. Infect. 62 (2006) 58. [6] J. Tian, K.K.Y. Wong, C.-M. Ho, C.-N. Lok, W.-Y. Yu, C.-M. Che, J.-F. Chiu, P.K.H. Tam, ChemMedChem 2 (2007) 129. [7] K. Galiano, C. Pleifer, K. Engelhardt, G. Brössner, P. Lackner, C. Huck, C. Lass-Flörl, A. Obwegeser, Neurol. Res. 30 (2008) 285. [8] D. Roe, B. Karandikar, N. Bonn-Savage, B. Gibbins, J.-B.J. Roullet, Antimicrob. Chemother. 61 (2008) 869. [9] P. Totaro, M. Rambaldini, Interact. Cardiovasc. Thorac. Surg. 8 (2009) 153. [10] H.J. Lee, S.H. Jeong, Text. Res. J. 75 (2005) 551. [11] S.T. Dubas, P. Kumlangdudsana, P. Potiyaraj, Colloids Surf. A 289 (2006) 105. [12] N.L. Lala, R. Ramaseshan, L. Bojun, S. Sundarrajan, R.S. Barhate, Y.-J. Liu, S. Ramakrishna, Biotechnol. Bioeng. 97 (2007) 1357. [13] T.M. Benn, P. Westerhoff, Environ. Sci. Technol. 42 (2008) 4133. [14] (a) H. Kong, J. Jang, Langmuir 24 (2008) 2051; (b) H. Kong, J. Jang, Biomacromolecules 9 (2008) 2677. [15] X. Xu, Q. Yang, J. Bai, T. Lu, Y. Li, X. Jing, J. Nanosci. Nanotechnol. 8 (2008) 5066. [16] C. Zhu, J. Xue, J. He, J. Nanosci. Nanotechnol. 9 (2009) 3067. [17] M. Wagener, Polym. Paint Colour J. 196 (2006) 34. [18] A. Kumar, P.K. Vemula, P.M. Ajayan, G. John, Nat. Mater. 7 (2008) 236. [19] J.-W. Rhim, S.-I. Hong, H.-M. Park, P.K.W. Ng, J. Agric. Food. Chem. 54 (2006) 5814. [20] R. Jung, Y. Kim, H.-S. Kim, H.-J. Jin, J. Biomater. Sci., Polym. Ed. 20 (2009) 311. [21] R. Tankhiwale, S.K. Bajpai, Colloids Surf. B 69 (2009) 164. [22] H. Huang, Q. Yuan, X. Yang, Colloids Surf. B 39 (2004) 31. [23] C.R.K. Rao, D.C. Trivedi, Synth. Met. 155 (2005) 324.

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